Compounds for the treatment of cancer

ABSTRACT

Methods and pharmaceutical compositions for inhibiting 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) and the treatment of cancer are described.

CROSS-REFERENCE TO RELATED APPLICATION

This application depends from and claims priority to U.S. ProvisionalApplication No: 62/152,239 filed Apr. 24, 2015, the entire contents ofwhich are incorporated herein by reference.

TECHNICAL FIELD

The presently disclosed subject matter relates to methods, compounds,and pharmaceutical compositions for the treatment of cancer. Moreparticularly, the presently disclosed subject matter relates to the useof compounds for the selective inhibition of6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4).

Methods of using said compounds to reduce glycolytic flux, reduce theproliferative capacity of a cell, reduce fructose-2,6-bisphosphate(F2,6BP), and to treat cancer are described.

BACKGROUND

Neoplastic cells preferentially utilize glycolysis to satisfy theirincreased needs for energy and biosynthetic precursors. The PFKFBenzymes (PFKFB 1-4) synthesize fructose-2,6-bisphosphate (F2,6BP).F2,6BP activates 6-phosphofructo-1-kinase (PFK-1), an essential controlpoint in the glycolytic pathway. Until recently, the PFKFB3 isozyme hasbeen considered the principal source of the increased F2,6BP observed incancer cells. However, new evidence indicates the co-expression ofseveral PFKFB isozymes in transformed and untransformed tissues, as wellas increased expression of the PFKFB4 isoform in several neoplastic celllines and in tumors.

Accordingly, there remains a need in the art for PFKFB4 inhibitors andmethods of using the same that can effectively be used to targetneoplastic cells, including the mechanisms within those cells thatrelate to the preferential use of the glycolytic pathway. Morespecifically, there remains a need in the art for small molecule PFKFB4inhibitors that can pharmacologically disrupt the kinase domain ofPFKFB4 and therefore decrease the glucose metabolism and growth of humancancers. Importantly, the PFKFB4 inhibitors should be selective forPFKFB4, and should not directly inhibit PFKFB1, PFKFB2, and PFKFB3.Furthermore, the PFKFB4 inhibitor should have good oral bioavailabilitywhile avoiding toxicity.

SUMMARY

Accordingly, the presently disclosed subject matter relates to the useof compounds for the selective inhibition of PFKFB4. Methods of usingsaid compounds to reduce glycolytic flux, reduce the proliferativecapacity of a cell, reduce fructose-2,6-bisphosphatc (F2,6BP), and totreat cancer are disclosed herein.

One embodiment of the presently-disclosed subject matter is directed toa method of treating cancer in a subject in need of treatment thereof,the method comprising administering to the subject an effective amountof a compound of:

wherein:R₁ is a C₁-C₅ alkyl nitrooxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

Another embodiment of the presently-disclosed subject matter is directedto a method of treating cancer in a subject in need of treatmentthereof, the method comprising administering to the subject an effectiveamount of a compound of:

wherein:n is 1-5;R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

One embodiment of the presently-disclosed subject matter is directed toa method of treating cancer in a subject in need of treatment thereof,the method comprising administering to the subject an effective amountof a compound of:

wherein:R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present, the nitrogen of the quinoline group has apositive charge.

A further embodiment of the presently-disclosed subject matter isdirected to a method of treating cancer in a subject in need oftreatment thereof, the method comprising administering to the subject aneffective amount of a compound of:

wherein:n is 1-6;R₁ is carboxylic acid, methyl sulfamide, carboxylic acid methyl ester,hydroxide, nitrate, or tert-butyl carbamate;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy,chloride, or hydrogen; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

Another embodiment of the presently-disclosed subject matter is directedto a method of treating cancer in a subject in need of treatmentthereof, the method comprising administering to the subject an effectiveamount of a compound of:

wherein:n is 1-6;R₁ nitrate; andR₂ is a hydrogen or nitrogen dioxide.

One embodiment of the presently-disclosed subject matter is directed toa method of treating cancer in a subject in need of treatment thereof,the method comprising administering to the subject an effective amountof a compound 5-[(8-methoxyquinolin-4-yl)amino]pentyl nitrate (5MPN).

An additional embodiment of the presently-disclosed subject matter isdirected to a method of treating cancer in a subject in need oftreatment thereof, the method comprising administering to the subject aneffective amount of a compound5-[(8-methoxyl-methylquinolin-1-ium-4-yl)amino]pentyl nitrate (MPN-2).

An additional embodiment is directed to a method of inhibiting PFKFB4 ina cell, the method comprising contacting the cell with an effectiveamount of a compound of:

wherein:R₁ is a C₁-C₅ alkyl nitrooxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

A further embodiment is directed to a method of inhibiting PFKFB4 in acell, the method comprising contacting the cell with an effective amountof a compound of:

wherein:n is 1-5;R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

An additional embodiment is directed to a method of inhibiting PFKFB4 ina cell, the method comprising contacting the cell with an effectiveamount of a compound of:

wherein:R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present, the nitrogen of the quinoline group has apositive charge.

A further embodiment of the presently-disclosed subject matter isdirected to a method of inhibiting PFKFB4 in a cell, the methodcomprising contacting the cell with an effective amount of a compoundof:

wherein:n is 1-6;R₁ is carboxylic acid, methyl sulfamide, carboxylic acid methyl ester,hydroxide, nitrate, or tert-butyl carbamate;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy,chloride, or hydrogen; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

Another embodiment of the presently-disclosed subject matter is directedto a method of inhibiting PFKFB4 in a cell, the method comprisingcontacting the cell with an effective amount of a compound of:

wherein:n is 1-6;R₁ nitrate; andR₂ is a hydrogen or nitrogen dioxide.

Another embodiment is directed to a method of inhibiting PFKFB4 in acell, the method comprising contacting the cell with an effective amountof 5MPN.

A further embodiment is directed to a method of inhibiting PFKFB4 in acell, the method comprising contacting the cell with an effective amountof MPN-2.

One embodiment is directed to a method of inhibiting PFKFB4 in asubject, the method comprising administering to the subject an effectiveamount of a compound of:

wherein:R₁ is a C₁-C₅ alkyl nitrooxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅, alkoxy; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

One embodiment is directed to a method of inhibiting PFKFB4 in asubject, the method comprising administering to the subject an effectiveamount of a compound of:

wherein:n is 1-5;R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

One embodiment is directed to a method of inhibiting PFKFB4 in asubject, the method comprising administering to the subject an effectiveamount of a compound of:

wherein:R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present, the nitrogen of the quinoline group has apositive charge.

A further embodiment of the presently-disclosed subject matter isdirected to a method of inhibiting PFKFB4 in a subject, the methodcomprising contacting the cell with an effective amount of a compoundof:

wherein:n is 1-6;R₁ is carboxylic acid, methyl sulfamide, carboxylic acid methyl ester,hydroxide, nitrate, or tert-butyl carbamate;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy,chloride, or hydrogen; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

Another embodiment of the presently-disclosed subject matter is directedto a method of inhibiting PFKFB4 in a subject, the method comprisingcontacting the cell with an effective amount of a compound of:

wherein:n is 1-6;R₁ nitrate; andR₂ is a hydrogen or nitrogen dioxide.

A further embodiment is directed to a method of inhibiting PFKFB4 in asubject, the method comprising administering to the subject an effectiveamount of 5MPN.

Another embodiment is directed to a method of inhibiting PFKFB4 in asubject, tire method comprising administering to the subject aneffective amount of MPN-2.

An additional embodiment is directed to a method of reducing glycolyticflux in a cell, the method comprising contacting the cell with aneffective amount of a compound of:

wherein:R₁ is a C₁-C₅ alkyl nitrooxy; R₂ can be present or absent, and whenpresent is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

One embodiment is directed to a method of reducing glycolytic flux in acell, the method comprising contacting the cell with an effective amountof a compound of:

wherein:n is 1-5;R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

An additional embodiment is directed to a method of reducing glycolyticflux in a cell, the method comprising contacting the cell with aneffective amount of a compound of:

wherein:R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present, the nitrogen of the quinoline group has apositive charge.

An additional embodiment is directed to a method of reducing glycolyticflux in a cell, the method comprising contacting the cell with aneffective amount of 5MPN.

One embodiment is directed to a method of reducing glycolytic flux in acell, the method comprising contacting the cell with an effective amountof MPN-2.

An additional embodiment is directed to a method of reducingproliferative capacity of a cell, the method comprising contacting thecell with an effective amount of a compound of:

wherein:R₁ is a C₁-C₅ alkyl nitrooxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

A further embodiment is directed to a method of reducing proliferativecapacity of a cell, the method comprising contacting the cell with aneffective amount of a compound of:

wherein:n is 1-5;R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

An additional embodiment is directed to a method of reducingproliferative capacity of a cell, the method comprising contacting thecell with an effective amount of a compound of:

wherein:R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present, the nitrogen of the quinoline group has apositive charge.

A further embodiment of the presently-disclosed subject matter isdirected to a method of reducing proliferative capacity of a cell, themethod comprising contacting the cell with an effective amount of acompound of:

wherein:n is 1-6;R₁ is carboxylic acid, methyl sulfamide, carboxylic acid methyl ester,hydroxide, nitrate, or tert-butyl carbamate;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy,chloride, or hydrogen; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

Another embodiment of the presently-disclosed subject matter is directedto a method of reducing proliferative capacity of a cell, the methodcomprising contacting the cell with an effective amount of a compoundof:

wherein:n is 1-6;R₁ nitrate; andR₂ is a hydrogen or nitrogen dioxide.

One embodiment is directed to a method of reducing proliferativecapacity of a cell, the method comprising contacting the cell with aneffective amount of 5MPN.

Another embodiment is directed to a method of reducing proliferativecapacity of a cell, the method comprising contacting the cell with aneffective amount of MPN-2.

An additional embodiment is directed to a method of reducingfructose-2,6-bisphosphate (F2,6BP) in a cell, the method comprisingcontacting the cell with an effective amount of a compound of:

wherein:R₁ is a C₁-C₅ alkyl nitrooxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

A further embodiment is directed to a method of reducingfructose-2,6-bisphosphate (F2,6BP) in a cell, the method comprisingcontacting the cell with an effective amount of a compound of:

wherein:n is 1-5;R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

An additional embodiment is directed to a method of reducingfructose-2,6-bisphosphate (F2,6BP) in a cell, the method comprisingcontacting the cell with an effective amount of a compound of:

wherein:R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present, the nitrogen of the quinoline group has apositive charge.

A further embodiment is directed to a method of reducingfructose-2,6-bisphosphate (F2,6BP) in a cell, the method comprisingcontacting the cell with an effective amount of 5MPN.

An additional embodiment is directed to a method of reducingfructose-2,6-bisphosphate (F2,6BP) in a cell, the method comprisingcontacting the cell with an effective amount of MPN-2.

An additional embodiment is directed to a pharmaceutical compositioncomprising an effective amount of a compound of:

wherein:R₁ is a C₁-C₅ alkyl nitrooxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge; andand at least one pharmaceutical excipient.

One embodiment is directed to a pharmaceutical composition comprising aneffective amount of a compound of:

wherein:n is 1-5;R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge; andand at least one pharmaceutical excipient.

An additional embodiment is directed to a pharmaceutical compositioncomprising an effective amount of a compound of:

wherein:R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present, the nitrogen of the quinoline group has apositive charge; and and at least one pharmaceutical excipient.

A further embodiment of the presently-disclosed subject matter isdirected to a pharmaceutical composition comprising an effective amountof a compound of:

wherein:n is 1-6;R₁ is carboxylic acid, methyl sulfamide, carboxylic acid methyl ester,hydroxide, nitrate, or tert-butyl carbamate;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy,chloride, or hydrogen; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge; andand at least one pharmaceutical excipient.

Another embodiment of the presently-disclosed subject matter is directedto a pharmaceutical composition comprising an effective amount of acompound of:

wherein:n is 1-6;R₁ nitrate; andR₂ is a hydrogen or nitrogen dioxide;and at least one pharmaceutical excipient.

An additional embodiment is directed to a pharmaceutical compositioncomprising an effective amount of 5MPN, and at least one pharmaceuticalexcipient.

A further embodiment is directed to a pharmaceutical compositioncomprising an effective amount of MPN-2, and at least one pharmaceuticalexcipient.

An additional embodiment of the presently-disclosed subject matter isdirected to a compound of Formula (IV):

wherein:n is 1-6;R₁ is carboxylic acid, methyl sulfamide, carboxylic acid methyl ester,hydroxide, nitrate, or tert-butyl carbamate;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy,chloride, or hydrogen; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

Another embodiment of the presently-disclosed subject matter is directedto a compound of Formula (V):

wherein:n is 1-6;R₁ nitrate; andR₂ is a hydrogen or nitrogen dioxide.

These and additional aspects and features of the instant invention willbe clarified by reference to the figures and detailed description setforth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Compound 5MPN inhibits recombinant PFKFB4 enzyme activity. FIG.1A shows a schematic representation of the 5MPN molecule docked in thecrystal structure of rat testes PFKFB4. 5MPN is shown in thicker stickrepresentation than the surrounding protein residues. FIG. 1B shows themolecular structure of 5MPN (MW, 305.3 kDa). FIG. 1C and FIG. 1D showMichaelis-Menlen and Line weaver-Burk double reciprocal plots,respectively, examining PFKFB4 enzyme activity as a function of F6Pconcentration (0-2000 μmol/L). In vitro kinase assays using purifiedrecombinant human PFKFB4 were performed as described in the presence orabsence of 0, 0.1, 1 or 10 μM 5MPN. Data shown are representative ofthree independent experiments.

FIG. 2. 5MPN causes decreased proliferation of cancer cells preceded bya reduction in intracellular F2,6BP concentration, glycolysis and ATP.FIG. 2A shows die results of H460 NSCLC cells treated with DMSO±10 μM5MPN. The effects on F2,6BP production, glycolysis and ATP were measuredafter 6-72 hours. FIG. 2B shows the proliferation of H460 cells exposedto DMSO±5MPN after 24-72 hours. FIG. 2C shows the cell growth of NHBEcells and indicated transformed cell lines that were exposed toDMSO±5MPN. Viable cells counted at 48 hours. FIG. 2D shows cell growthof NHBE and hT/LT/Ras cells treated with DMSO±5MPN. Live cells werecounted at 48 hours. (*p value <0.01 hT/LT/Ras vs. NHBE). FIG. 2E, showsPFKFB4 expression of H460 cells transfected with empty pCMV-XL4 (Vec) orpCMV-XL4 containing full-length PFKFB4 (FB4) for 24 hours that weretreated with DMSO±10 μM 5MPN. 24 hours after treatment with DMSO±10 μM5MPN, PFKFB4 expression was examined by Western blot and viable cellswere counted. (*p value <0.01 Vec vs. FB4 exposed to 5MPN). FIG. 2Fshows large T antigen-immortalized, tamoxifen (4HT)-induciblePFKFB4^(−/−) lung fibroblasts that were exposed to vehicle (ethanol) ±10μM 4HT for 24 hours and then treated with DMSO±10 μM 5MPN. Cell countsand PFKFB4 protein expression were examined 24 hours later. (*p value<0.01 vehicle vs. +4HT, exposed to 5MPN). Data are expressed as themean±SD of three experiments.

FIG. 3. 5MPN induces cell cycle arrest at the G1 phase. FIG. 3A showsH460 cells treated with DMSO±10 μM 5MPN, while FIG. 3B shows H460 cellstreated transfected with nonsense (siCtrl) or PFKFB4 siRNA (siFB4).Cells were analyzed for induction of apoptosis by flow cytometry.Decrease in PFKFB4 protein expression by siFB4 was confirmed by Westernblot. PI⁺+PI/Ann V⁺ cells shown as % apoptotic cells. FIG. 3C shows H460cells treated with DMSO±10 μM 5MPN and the distribution of cells in G1,S and G2 phases of the cell cycle. FIG. 3D shows H460 cells transfectedwith siCtrl or siFB4 and the distribution of cells in phases of the cellcycle, H460 cells were transfected with empty pCMV-XL4 (Vec) or pCMV-XL4containing PFKFB4 (FB4) for 24 hours then treated with DMSO±10 μM 5MPNfor 24 hours. FIG. 3E shows H460 cells transfected with siCtrl or siFB4and PFKFB4 protein expression and F2,6BP concentration, H460 cells weretransfected with empty pCMV-XL4 (Vec) or pCMV-XL4 containing PFKFB4(FB4) for 24 hours then treated with DMSO±10 μM 5MPN for 24 hours. (*pvalue <0.01 Vec vs. FB4 exposed to 5MPN.) FIG. 3F shows H460 cellstransfected with siCtrl or siFB4 and the distribution of cells in G1, Sand G2 phases of the cell cycle. Data shown are representative of threeindependent experiments and are expressed as the mean±SD of threeexperiments. *p value <0.01 compared to control.

FIG. 4. 5MPN has high oral bioavailability and suppresses glucose uptakeand tumor growth in mice. FIG. 4A and FIG. 4B show the oral andintravenous pharmacokinetic properties of 5MPN were in C57BL/6 mice.Groups of 10 C57BL/6 mice were implanted with LLC cells and, when tumorsreached a mass of 150-200 mg, were randomized to daily administration ofDMSO or 5MPN by gavage (120 mg/kg, for two weeks). FIG. 4C and FIG. 4Dshow daily tumor and daily body mass measurements, respectively. FIG. 4Eshows F2,6BP expression after 10 days of daily administration of DMSO or5MPN (120 mg/kg) by gavage. The mice were euthanized, and tumorsextracted and analyzed for F2,6BP (shown as % of DMSO). FIG. 4F showsmicro-PET scans obtains from separate groups of tumor-bearing mice thatwere administered either DMSO or 5MPN (120 mg/kg by gavage, once).Regions of interest in the tumor and cerebellum were quantified inquadruplicate. Representative transverse view cuts are shown with redarrows indicating the tumor. FIG. 4G shows the in vitro cell cycleanalysis of LLC cells that were exposed to DMSO±10 μM 5MPN through theexamination of Ki67-positive cells. FIG. 4H shows Ki67 staining in LLCxenographs that were examined by immunohistochcmistry (representativesections shown. 10× and 25× magnification). Groups of 10 C57BL/6 micewere implanted with LLC cells and, when tumors reached a mass of 150-200mg, were randomized to daily administration of DMSO or 5MPN by gavage(120 mg/kg, for two weeks). FIG. 4I shows Ki67 positive pixels that wereenumerated in a minimum of 5 Helds per tumor section. Groups of 10C57BL/6 mice were implanted with LLC cells and, when tumors reached amass of 150-200 mg, were randomized to daily administration of DMSO or5MPN by gavage (120 mg/kg, for two weeks). FIG. 4J and FIG. 4K showtumor and body mass measurements (collected daily), respectively, ofgroups of 10 BALB/c athymic mice implanted with H460 NSCLC cells and,when tumors were 150-200 mg, were randomized to daily DMSO or 5MPN bygavage. *p value <0.01 compared to controls.

FIG. 5. MPN-2 inhibits recombinant PFKFB4 enzyme activity, decreases theproduction of F2,6BP, decreased proliferation of cancer cells, and hashigh oral bioavailability.

FIG. 5A shows the molecular structure of MPN-2. FIG. 5B shows that MPN-2significantly inhibits PFK.FB4 activity. FIG. 5C shows that MPN-2significantly inhibits F2,6BP production. FIG. 5D shows that MPN-2decreases the proliferation of a human cancer cell line, H460 NSCLCcells. Viable cells counted at 48 hours and at 72 hours. FIG. 5E showsthe oral and intravenous pharmacokinetic properties of MPN-2 in CD-1mice (N=3).

FIG. 6. MPN-2 causes decreased proliferation of cancer cells preceded bya reduction in intracellular glycolysis and ATP, decreased proliferationof cancer cells, and tumor growth in mice. FIG. 6A and FIG. 6B shows theresults of H460 NSCLC cells treated with DMSO±the indicatedconcentrations of MPN-2. The effects on glycolysis (FIG. 6A) and ATP(FIG. 6B) were measured after 48 hours. FIG. 6C shows that MPN-2decreases the proliferation of MCF7, SK-BR-3, H460, and A549 cellsexposed to DMSO±the indicated concentrations of MPN-2 after 24-72 hours(72 hours shown). FIG. 6D show daily tumor mass measurements of groupsof 10 C57BL/6 mice that were implanted with LLC cells and, when tumorsreached a mass of 150-200 mg, were randomized to daily administration ofDMSO or 5MPN in DMSO at the indicated dose by intraperitoneal injection(10 mg/kg, for eleven days).

FIG. 7. Formula IV compounds decreased proliferation of cancer cells.FIG. 7 shows the results of H460 NSCLC cells treated with DMSO±theindicated concentrations of two compounds of Formula IV at the indicateddoses. Specifically,

FBR1-02 corresponds to

while FBR1-09 corresponds to

FIG. 8. Dual PFKFB4 and PFKFB3 inhibition with MPN-2 and PFK15,respectively, causes a synergistic increase in ceil death. FIG. 8A showsthe Fa-Cl combination index plot, while FIG. 8B shows the isobologram.

FIG. 9. Synthesis pathway of the small molecule antagonists of thekinase domain of PFKFB4. FIG. 9 shows a synthesis pathway used to createvarious compounds of Formulae (I), (II), and (III), (IV), (V), (VI), and(VII)).

DETAILED DESCRIPTION

Particular details of various embodiments of the invention are set forthto illustrate certain aspects and not to limit the scope of theinvention. It will be apparent to one of ordinary skill in the art thatmodifications and variations are possible without departing from thescope of the embodiments defined in the appended claims. Morespecifically, although some aspects of embodiments of the presentinvention may be identified herein as preferred or particularlyadvantageous, it is contemplated that the embodiments of the presentinvention are not limited to these preferred aspects.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.

The presently disclosed data demonstrates that highly specific smallmolecule antagonists of the kinase domain of PFKFB4 suppress glucosemetabolism and the proliferation of multiple cancer types. Importantly,such molecules reduce the glycolysis and intracellular F2,6BP of cancercells, but do not inhibit recombinant PFK-1 or PFKFB3, which share thesame substrate-binding domain and are also expressed in multiple cancercell lines. Furthermore, the presently-disclosed data demonstrates thatthe cell cycle arrest effects of the small molecule antagonists of thekinase domain of PFKFB4 can be overcome by over-expression of PFKFB4,indicating that the anti cancer effects of such small moleculeantagonists are due, at least in part, to their inhibition of PFKFB4.Because these small molecule antagonists of the kinase domain of PFKFB4suppress glycolytic flux through the enolase reaction, the availabilityof both fructose 6-phosphate and glyceraldehyde 3-phosphate for ribosesynthesis via the non-oxidative pentose shunt is reduced by these smallmolecule antagonists. Accordingly, the presently-disclosed dataindicates that the observed G1 arrest in vitro caused by thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (and by PFKFB4 siRNA) is a direct result of reduced availabilityof these glycolytic intermediates that are required for DNA synthesisduring the S phase.

Molecular modeling was used to conduct virtual screens for novel ligandsthat might bind to the kinase as opposed to the bisphosphatase domain,due to the kinase activity being essential for neoplastic glucosemetabolism and growth. The instant data demonstrates that thepresently-disclosed small molecules are the first small moleculeantagonists of PFKFB4 kinase activity. The presently-disclosed datademonstrates that said molecules not only reduce F2,6BP, but alsoglycolytic flux through PFK-1 and cell cycle progression into the Sphase. The observation that pharmacological inhibition of the kinasedomain of PFKFB4 suppresses cell proliferation thus provides“proof-of-concept” that PFKFB4 kinase inhibitors, as opposed tobisphosphatase inhibitors, may have utility as anti-cancer agents.

A related family member, PFKFB3, is encoded on a different chromosomeand has a kinase:phosphatase ratio and tissue distribution distinct fromPFKFB4. A series of small molecules have been developed that selectivelyinhibit PFKFB3. The relative roles of PFKFB3 and PFKFB4 are poorlyunderstood. However, PFKFB4 appears to be essential for cancer cellsurvival and correlates highly with hypoxic regions of tumors, whereasPFKFB3 localizes to both the cytoplasm and the nucleus where itactivates PFK-1 and cyclin dependent kinase 1 respectively.Surprisingly, the instant disclosure demonstrates that these two enzymesmay provide some degree of reciprocal compensation and that dualinhibition of PFKFB3 and PFKFB4 may yield optimal suppression ofintracellular F2,6BP and cell viability. In fact, studies conducted bythe instant investigators have revealed that PFKFB4 expression isincreased by PFKFB3 inhibition, suggesting that PFKFB4 may compensatefor decreased PFKFB3 expression and activity and, importantly, may limitthe efficacy of PFK15 (Tocris) and other PFKFB3 inhibitors. Theinstantly-disclosed data demonstrates that simultaneous administrationof MPN-2 (determined by the instant investigators to be a PFKFB4inhibitor) and PFK15 (Tocris, a commercially available PFKFB3 inhibitor)synergistically increases cell death in vitro. Thus, combination therapywith PFK15 (and other PFKFB3 inhibitors) and the presently-disclosedsmall molecule antagonists of the kinase domain of PFKFB4 may be used toprovide an effective chemotherapeutic regimen.

Although there has been some degree of trepidation regarding thepharmacological targeting of enzymes that regulate an essentialbiochemical process such as glycolysis, the presently disclosed datademonstrate that PFKFB4, an enzyme expressed in several normal organs,can be pharmacologically inhibited without gross, histological, orlaboratory signs of toxicity. Furthermore, the instant data demonstratethat the presently-disclosed small molecule inhibitors of the kinasedomain of PFKFB4 were selectively cytostatic to RAS-transformed cellsand not normal cells, and suppressed tumor growth without causingtoxicity. This data is at least consistent with the hypothesis thatneoplastic cells may be metabolically reprogrammed to rely more heavilyon this regulator of metabolism. It is noteworthy that an inhibitor ofthe essential non-mutated cell cycle regulatory enzymes, CDK4 and CDK6,palbocicib, was recently found to double the progression-free survivalof breast cancer patients without causing excessive toxicity. Based onthis recent clinical success and the presently-disclosed data, PFKFB4inhibitors that suppress glucose metabolism, such as thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4, may yield favorable therapeutic indices in patients sufferingwith advanced solid cancers.

Accordingly, the presently-disclosed subject matter includes a method oftreating cancer in a subject in need of treatment comprisesadministering to the subject an effective amount of a compound havingthe Formula (I):

wherein:R₁ is a C₁-C₅ alkyl nitrooxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

In some embodiments, a method of treating cancer in a subject in need oftreatment comprises administering to the subject an effective amount ofa compound having the Formula (II):

wherein:n is 1-5;R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge.

In other embodiments, a method of treating cancer in a subject in needof treatment comprises administering to the subject an effective amountof a compound having the Formula (III):

wherein:R₁ can be present or absent, and when present is a C₁-C₅ alkoxy;R₂ can be present or absent, and when present is a C₁-C₅ alkyl; andwherein if R₂ is present, the nitrogen of the quinoline group has apositive charge.

In further embodiments, a method of treating cancer in a subject in needof treatment thereof comprises administering to the subject an effectiveamount of a compound of:

wherein:n is 1-6;R₁ is carboxylic acid, methyl sulfamide, carboxylic acid methyl ester,hydroxide, nitrate, or tert-butyl carbamate;R₂ can be present or absent, and when present is a C₁-C₅ alkyl;R₃ can be present or absent, and when present is a C₁-C₅ alkoxy, C₁-C₅alkyl, chlorine, or hydrogen; andwherein if R₂ is present and located on the nitrogen of the quinolinegroup, said nitrogen has a positive charge. In other embodiments, R1 isa carbonate, a carbamide, nitro sulfonamide, or a thiocarbonyl. Incertain embodiments, the compound of formula IV includes the compoundshaving the following formulae:

In other embodiments, a method of treating cancer in a subject in needof treatment thereof comprises administering to the subject an effectiveamount of a compound of:

wherein:n is 1-6;R₁ nitrate; andR₂ is a hydrogen or nitrogen dioxide. In further embodiments, R1 iscarboxylic acid, methyl sulfamide, carboxylic acid methyl ester,hydroxide, nitrate, or tert-butyl carbamate. In even furtherembodiments, R1 is a carbonate, a carbamide, nitro sulfonamide, or athiocarbonyl. In certain embodiments, the compound of formula V includesthe compounds having the following formulae:

In additional embodiments, a method of treating cancer in a subject inneed of treatment thereof comprises administering to the subject aneffective amount of 5MPN.

In further embodiments, a method of treating cancer in a subject in needof treatment thereof comprises administering to the subject an effectiveamount of MPN-2.

In some embodiments, a method of treating cancer in a subject in need oftreatment thereof comprises an effective amount of compound:

wherein:n is 2-6;R1 can be present or absent, and when present is a C₁-C₅ alkoxy, a C₁-C₅alkyl, chlorine, or hydrogen;R₂ can be present or absent, and when present is a hydrogen or nitrogendioxide;W is carbon, oxygen, nitrogen, hydrogen, methyl, methoxy, or hydroxide;X can be present or absent, and when present is carbon, oxygen,nitrogen, or sulfer;Y can be present or absent, and when present is oxygen, sulfer, orindependently oxygen and sulfer and wherein one or two Ys may bepresent; andZ is can be present or absent, and when present is carbon, oxygen,nitrogen, hydrogen, methyl, or methoxy; andwherein if W is hydrogen, methyl, methoxy, or hydroxide, then X, Y, andZ is absent.

In some embodiments, a method of treating cancer in a subject in need oftreatment thereof comprises an effective amount of compound:

wherein:n is 1-6;R₂ is a hydrogen or nitrogen dioxide;W is carbon, oxygen, nitrogen, hydrogen, methyl, methoxy, or hydroxide;X can be present or absent, and when present is carbon, oxygen,nitrogen, or sulfer;Y can be present or absent, and when present is oxygen, sulfer, orindependently oxygen and sulfer and wherein one or two Ys may bepresent; andZ is can be present or absent, and when present is carbon, oxygen,nitrogen, hydrogen, methyl, or methoxy; andwherein if W is hydrogen, methyl, methoxy, or hydroxide, then X, Y, andZ is absent.

“Cancer” refers to diseases caused by uncontrolled cell division and theability of cells to metastasize, or to establish new growth inadditional sites. It will be appreciated that the administration to asubject of an effective amount of the presently-disclosed small moleculeantagonists of the kinase domain of PFKFB4 can provide therapy for awide variety of cancers including, but not limited to solid tumors, suchas lung, breast, colon, ovarian, brain, liver, pancreas, prostate,malignant melanoma, non-melanoma skin cancers, as well as hematologictumors and/or malignancies, such as childhood leukemia and lymphomas,multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic andcutaneous origin, acute and chronic leukemia such as acutelymphoblastic, acute myelocytic or chronic myelocytic leukemia, plasmacell neoplasm, lymphoid neoplasm and cancers associated with AIDS. Incertain embodiments of a method of treating cancer in a subject in needof treatment thereof comprising administering to the subject aneffective amount of the presently-disclosed small molecule antagonistsof the kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2),the cancer is selected from breast cancer, lung cancer, colon cancer,and prostate cancer.

As used herein, the term “treating” relates to any treatment of cancer,including but not limited to prophylactic treatment and therapeutictreatment. “Treating” includes any effect, e.g., lessening, reducing,modulating, or eliminating, that results in the improvement of thecancer. “Treating” or “treatment” of cancer state includes: inhibitingthe cancer, i.e., arresting the development of the cancer or itsclinical symptoms; or relieving the cancer, i.e., causing temporary orpermanent regression of the cancer or its clinical symptoms.

A “subject” includes mammals, e.g., humans, companion animals (e.g.,dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs,horses, fowl, and the like) and laboratory animals (e.g., rats, mice,guinea pigs, birds, and the like). In certain embodiments of a method oftreating cancer in a subject in need of treatment thereof comprisingadministering to the subject an effective amount of thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2), the subject that isadministered an effective amount is a mammal.

An “effective amount” is defined herein in relation to the treatment ofcancers is an amount that will decrease, reduce, inhibit, or otherwiseabrogate the growth of a cancer cell or tumor. The “effective amount”will vary depending the cancer and its severity and the age, weight,etc., of the mammal to be treated.

It will be understood that the presently-disclosed small moleculeantagonists of the kinase domain of PFKFB4 (including the compounds ofFormulae (I), (II), and (III), (IV), (V), (VI), and (VII) as well as5MPN or MPN-2), can include pharmaceutically acceptable salts, solvates,stereoisomers, and optical isomers thereof. It will further beunderstood that the compounds of Formulae (I), (II), and (ID), (IV),(V), (VI), and (VII), as well as 5MPN or MPN-2, can include prodrugs ofsuch compounds.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. As used herein,“pharmaceutically acceptable salt” refers to derivative of the compoundsof Formulae (I), (II), and (III), (IV), (V), (VI), and (VII) as well as5MPN or MPN-2, wherein such compounds are modified by making acid orbase salts thereof. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines, alkali or organic salts of acidic residues suchas carboxylic acids, and the like. The pharmaceutically acceptable saltsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. For example, such conventional non-toxicsalts include, but are not limited to, those derived from inorganic andorganic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic,acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic,citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric,glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic,hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic,hydroxymalcic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric,oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic,propionic, salicyclic, stearic, subacetic, succinic, sulfamic,sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and thecommonly occurring amine acids, e.g., glycine, alanine, phenylalanine,arginine, etc.

It should be understood that all references to the presently-disclosedsmall molecule antagonists of the kinase domain of PFKFB4 (including thecompounds of Formulae (I), (II), and (III), (IV), (V), (VI), and (VII)as well as 5MPN or MPN-2), or pharmaceutically acceptable salts thereof,include solvent addition forms (solvates) or crystal forms (polymorphs)as defined herein.

The terms “crystal polymorphs” or “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or salt or solvate thereof) cancrystallize in different crystal packing arrangements, all of which havethe same elemental composition. Different crystal forms usually havedifferent X-ray diffraction patterns, infrared spectral, melting points,density hardness, crystal shape, optical and electrical properties,stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

Additionally, the compounds of the present invention, for example, thesalts of the presently-disclosed small molecule antagonists of thekinase domain of PFKFB4 (including the compounds of Formulae (I), (II),and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2), canexist in either hydrated or unhydrated (the anhydrous) form or assolvates with other solvent molecules. Nonlimiting examples of hydratesinclude monohydrates, dihydrates, etc. Nonlimiting examples of solvatesinclude ethanol solvates, acetone solvates, etc.

“Solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundsor salts have a tendency to trap a fixed molar ratio of solventmolecules in the crystalline solid state, thus forming a solvate. If thesolvent is water the solvate formed is a hydrate, when the solvent isalcohol, the solvate formed is an alcoholate. Hydrates are formed by thecombination of one or more molecules of water with one of the substancesin which the water retains its molecular slate as H₂O, such combinationbeing able to form one or more hydrate.

The presently-disclosed small molecule antagonists of the kinase domainof PFKFB4 (including the compounds of Formulae (I), (II), and (III),(IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) of the presentinvention can be prepared as prodrugs, for example pharmaceuticallyacceptable prodrugs. The terms “pro-drug” and “prodrug” are usedinterchangeably herein and refer to any compound which releases anactive parent drug in vivo. Since prodrugs are known to enhance numerousdesirable qualities of pharmaceuticals (e.g., solubility,bioavailability, manufacturing, etc.) The presently-disclosed smallmolecule antagonists of the kinase domain of PFKFB4 (including thecompounds of Formulae (I), (II), and (III), (IV), (V), (VI), and (VII)as well as 5MPN or MPN-2) can be delivered in prodrug form. Thus, thepresent invention is intended to cover prodrugs of thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2), methods of deliveringthe same and compositions containing the same. “Prodrugs” are intendedto include any covalently bonded carriers that release an active parentdrug of the present invention in vivo when such prodrug is administeredto a subject.

In certain embodiments, the presently-disclosed small moleculeantagonists of the kinase domain of PFKFB4 (including the compounds ofFormulae (I), (II), and (III), (IV), (V), (VI), and (VII) as well as5MPN or MPN-2) are administered at a dosage effective for specificallyinhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphophatase 4(PFKFB4). In more specific embodiments, “specifically inhibiting” isdefined as inhibiting PFKFB4 without inhibiting6-phosphofructo-2-kinase/fructose-2,6,biphosphate 3 (PFKFB3),6-phosphofructo-2-kinase/fructose-2,6-biphospatase 2 (PFKFB2), or6-phosphofructo-2-kinase/fructose-2,6,biphosphate 1 (PFKFB1).

In some embodiments, the presently-disclosed small molecule antagonistsof the kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (111), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2)can be delivered regionally to a particular affected region or regionsof the subject's body. In some embodiments, the presently-disclosedsmall molecule antagonists of the kinase domain of PFKFB4 (including thecompounds of Formulae (I), (II), and (III), (IV), (V), (VI), and (VII)as well as 5MPN or MPN-2) can be administered systemically. For example,in some embodiments of a method treating cancer in a subject in need oftreatment thereof, the presently-disclosed small molecule antagonists ofthe kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2)are administered orally. In accordance with the presently disclosedmethods, the presently-disclosed small molecule antagonists of thekinase domain of PFKFB4 (including the compounds of Formulae (I), (II),and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) can beadministered orally as a solid or as a liquid. In other embodiments oftreating cancer in a subject in need of treatment, thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2) is administeredintravenously. In accordance with the presently disclosed methods, thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2) can be administeredintravenously as a solution, suspension, or emulsion. Alternatively, thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (IT), and (III), (VI),(V), (VI), and (VII) as well as 5MPN or MPN-2) also can be administeredby inhalation, intravenously, or intramuscularly as a liposomalsuspension.

In certain embodiments of a method of treating cancer in a subject inneed of treatment thereof comprising administering to the subject aneffective amount of the presently-disclosed small molecule antagonistsof the kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2),the subject that is administered an effective amount of said smallmolecule antagonist is substantially free of signs of toxicity.“Substantially free of signs of toxicity” includes unsafe deviations oncomplete blood counts, electrolytes, hepatic and renal function, bodymass, and the unsafe deviations on the gross and histological appearanceof the brain, heart, lungs, liver, kidneys, and spleen due to theadministration of the presently-disclosed small molecule antagonists ofthe kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-22)to the subject.

In some embodiments of a method of treating cancer in a subject in needof treatment thereof comprising administering to the subject aneffective amount of the presently-disclosed small molecule antagonistsof the kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2),the method further comprises administering to the subject one or moreadditional therapeutic compounds. It will be appreciated thattherapeutic benefits for the treatment of cancer can be realized bycombining treatment with the presently-disclosed small moleculeantagonists of the kinase domain of PFKFB4 (including the compounds ofFormulae (I), (II), and (III), (VI), (V), (VI), and (VII) as well as5MPN or MPN-2) with one or more additional therapeutic compounds. Theterm “additional therapeutic compounds” includes anti-cancer agents ortreatments. The choice of such combinations will depend on variousfactors including, but not limited to, the type of disease, the age andgeneral health of the subject, the aggressiveness of diseaseprogression, and the ability of the subject to tolerate the agents thatcomprise the combination. For example, the presently-disclosed smallmolecule antagonists of the kinase domain of PFKFB4 (including thecompounds of Formulae (I), (IT), and (III), (IV), (V), (VI), and (VII)as well as 5MPN or MPN-2) can be combined with other agents andtherapeutic regimens that are effective at reducing tumor size (e.g.,radiation, surgery, chemotherapy, hormonal treatments, and or genetherapy). Further, in some embodiments, it can be desirable to combinethe presently-disclosed small molecule antagonists of the kinase domainof PFKFB4 (including the compounds of Formulae (I), (II), and (III),(IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) with one or moreagents that treat the side effects of a disease or the side effects ofone of the additional therapeutic agents, e.g., providing the subjectwith an analgesic, or agents effective to stimulate the subject's ownimmune response (e.g., colony stimulating factor).

Thus, the term “additional therapeutic compounds” includes a variety ofinclude anti-cancer agents or treatments, such as chemical compoundsthat are also known as anti-neoplastic agents or chemotherapeuticagents. The agents can be used in combination with thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2). Such compounds include,but are not limited to, alkylating agents, DNA intercalators, proteinsynthesis inhibitors, inhibitors of DNA or RNA synthesis, DNA baseanalogs, topoisomerase inhibitors, anti-angiogenesis agents, andtelomerase inhibitors or telomeric DNA binding compounds. For example,suitable alkylating agents include alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as a benzodizepa,carboquone, meturedepa, and uredepa; ethylenimines and methylmelamines,such as altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cyclophosphamide, estramustine,iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichine, phenesterine, prednimustine, trofosfamide, anduracil mustard; nitroso ureas, such as carmustine, chlorozotocin,fotemustinc, lomustine, nimustine, and ranimustine.

Antibiotics used in the treatment of cancer and that can be combinedwith the presently-disclosed small molecule antagonists of the kinasedomain of PFKFB4 (including the compounds of Formulae (I), (II), and(in), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) includedactinomycin, daunorubicin, doxorubicin, idarubicin, bleomycin sulfate,mytomycin, plicamycin, and streptozocin. Chemotherapeuticantimetabolites can also be combined with the presently-disclosed smallmolecule antagonists of the kinase domain of PFKFB4 (including thecompounds of Formulae (I), (II), and (III), (IV), (V), (VI), and (VII)as well as 5MPN or MPN-2) for the treatment of cancer, and includemercaptopurine, thioguanine, cladribine, fludarabine phosphate,fluorouracil (5-FU), floxuridine, cytarabine, pentostatin, methotrexate,and azathioprine, acyclovir, adenine β-1-D-arabinoside, amethopterin,aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine,6-azauracil, 2′-azido-2′-deoxynucleosides, 5-bromodeoxycytidine,cytosine β-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides,5-fluorodeoxycytidine, 5-fluorodeoxyuridine, and hydroxyurea.

Chemotherapeutic protein synthesis inhibitors can also be combined withthe presently-disclosed small molecule antagonists of the kinase domainof PFKFB4 (including the compounds of Formulae (I), (II), and (III),(IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) for the treatmentof cancer. Such inhibitors include abrin, aurintricarboxylic acid,chloramphenicol, colicin E3, cyeloheximide, diphtheria toxin, edeine A,emetine, erythromycin, ethionine, fluoride, 5-fluorotryptophan, fusidicacid, guanylyl methylene diphosphonate and guanylyl imidodiphosphate,kanamycin, kasugamycin, kirromycin, and O-methyl threonine.

Additionally, protein synthesis inhibitors can also be combined with thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2) for the treatment ofcancer. Such inhibitors include modeccin, neomycin, norvaline,pactamycin, paromomycine, puromycin, ricin, shiga toxin, showdomycin,sparsomycin, spectinomycin, streptomycin, tetracycline, thiostrepton,and trimethoprim. Furthermore, inhibitors of DNA synthesis can becombined with the presently-disclosed small molecule antagonists of thekinase domain of PFKFB4 (including the compounds of Formulae (I), (II),and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) for thetreatment of cancer. Such inhibitors include alkylating agents such asdimethyl sulfate, mitomycin C, nitrogen and sulfur mustards,intercalating agents, such as acridine dyes, actinomycins, adriamycin,anthracenes, benzopyrene, ethidium bromide, propidiumdiiodide-intertwining, and agents, such as distamycin and netropsin.Topoisomerase inhibitors, such as coumermycin, nalidixic acid,novobiocin, and oxolinic acid, inhibitors of cell division, includingcolcemide, colchicine, vinblastine, and vincristine; and RNA synthesisinhibitors including actinomycin D, α-amanitine and other fungalamatoxins, cordycepin (3′-deoxyadenosine), dichlororibofuranosylbenzimidazole, rifampicine, streptovaricin, and streptolydigin also canbe combined with the presently-disclosed small molecule antagonists ofthe kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) toprovide a suitable cancer treatment.

Thus, current chemotherapeutic agents that can be used in a combinationtreatment with the presently-disclosed small molecule antagonists of thekinase domain of PFKFB4 (including the compounds of Formulae (I), (II),and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) include,but are not limited to, adrimycin, 5-fluorouracil (5FU), etoposide,camptothecin, actinomycin-D, mitomycin, cisplatin, hydrogen peroxide,carboplatin, procarbazine, mechlorethamine, cyclophosphamide,ifosfamide, melphalan, chjlorambucil, bisulfan, nitrosurea,dactinomycin, duanorubicin, doxorubicin, bleomycin, plicomycin,tamoxifen, taxol, transplatimun, vinblastin, and methotrexate, and thelike.

“Additional therapeutic compounds” can further involve immunotherapydirected at tumor antigen markers that are found on the surface of tumorcells. Treatment of a cancer with the presently-disclosed small moleculeantagonists of the kinase domain of PFKFB4 (including the compounds ofFormulae (I), (II), and (III), (IV), (V), (VI), and (VII) as well as5MPN or MPN-2) can further be combined with a gene therapy basedtreatment, targeted towards oncogenes and/or cell cycle controllinggenes, such as p53, p16, p21, Rb, APC, DCC, NF-1, NF-2, BRCA2, FHIT,WT-1, MEN-I, MEN U, BRCA1. VHL, FCC, MCC, ras, myc, neu, raf, erb, src,fms, jun, irk, ret, gsp, hst, bel, and abl, which are often mutatedversions of their normal cellular couterparts in cancerous tissues.

In more specific embodiments of a method of treating cancer in a subjectin need of treatment thereof comprising administering to the subject aneffective amount of the presently-disclosed small molecule antagonistsof the kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (VI), (V), (VI), and (VII) as well as 5MPN or MPN-2),one or more additional therapeutic compounds comprise one or more of aPFKFB3 inhibitor, a PFKFB2 inhibitor, and a PFKFB1 inhibitor. In certainembodiments, the one or more PFKFB3 inhibitor is PFK15.

The additional therapeutic agents can be administered by the same routeor by different routes. For example, a first therapeutic agent of thecombination selected may be administered by intravenous injection whilethe other therapeutic agents of the combination may be administeredorally. Alternatively, for example, all therapeutic agents may beadministered orally or all therapeutic agents may be administered byintravenous injection. The sequence in which the therapeutic agents areadministered is not narrowly critical.

Additional cancer treatments can be used in combination withadministration of the presently-disclosed small molecule antagonists ofthe kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (VI), (V), (VI), and (VII) as well as 5MPN or MPN-2).For example, the presently-disclosed small molecule antagonists of thekinase domain of PFKFB4 (including the compounds of Formulae (I), (II),and (III), (VI), (V), (VI), and (VII) as well as 5MPN or MPN-2) can beused as part of a treatment course further involving attempts tosurgically remove part or all of a cancerous growth. For instance, thepresently-disclosed small molecule antagonists of tire kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2) can be administered aftersurgical treatment of a subject to treat any remaining neoplastic ormetastasized cells. Treatment with the presently-disclosed smallmolecule antagonists of the kinase domain of PFKFB4 (including thecompounds of Formulae (I), (II), and (III), (IV), (V), (VI), and (VII)as well as 5MPN or MPN-2) can also precede surgery, in an effort toshrink the size of a tumor to reduce the amount of tissue to be excised,thereby making the surgery less invasive and traumatic. Furthermore,treatment with the presently-disclosed small molecule antagonists of thekinase domain of PFKFB4 (including the compounds of Formulae (I), (II),and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) caninclude one or more treatment courses with a radiotherapeutic agent toinduce DNA damage. Radiotherapeutic agents, include, for example, gammairradiation, X-rays. UV-irradiation, microwaves, electronic emissions,radioisotopes, and the like. Therapy can be achieved by irradiating thelocalized tumor site with the above-described forms of radiation.

In other embodiments of the presently-disclosed subject matter, apharmaceutical composition comprising an effective amount of thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2) and at least onepharmaceutical excipient is provided. “Pharmaceutically acceptableexcipient” means an excipient that is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable, and includes excipient that isacceptable for veterinary use as well as human pharmaceutical use. Thus,the term “pharmaceutical excipient” is used herein to describe anyingredient other than the compound(s) of the invention. Examples ofpharmaceutical excipients include one or more substances which may actas diluents, flavoring agents, solubilisers, lubricants, suspendingagents, binders, preservatives, wetting agents, tablet disintegratingagents, or an encapsulating material. The choice of excipient will to alarge extent depend on factors such as the particular mode ofadministration, the effect of the excipient on solubility and stability,and the nature of the dosage form. A “pharmaceutical excipient” includesboth one and more than one such excipient.

In certain embodiments of a pharmaceutical composition comprising aneffective amount of the presently-disclosed small molecule antagonistsof the kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2)and at least one pharmaceutical excipient, the presently-disclosed smallmolecule antagonists of the kinase domain of PFKFB4 (including thecompounds of Formulae (I), (II), and (III), (IV), (V), (VI), and (VII)as well as 5MPN or MPN-2) is an amount that specifically inhibitsPFKFB4. Thus, in embodiments of the pharmaceutical composition, thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 are specific inhibitors of the activity of PFKFB4. In someaspects, the specific inhibitors of PFKFB4 inhibits the activity ofPFKFB4 by at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%. 75%, 80%, 85%, 90%, 95% 99%, or any value or range in between, butdoes not inhibit the activity of PFKFB1, PFKFB2, PFKFB3 by more thanabout 20%, 15%, 10%, 5%, 2%, or 1%.

The pharmaceutical composition comprising an effective amount of thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2) and at least onepharmaceutical excipient described herein can be formulated with apharmaceutically acceptable carrier for administration to a human or ananimal. As such, the pharmaceutical compositions can be administeredorally as a solid or as a liquid, or can be administered intramuscularlyor intravenously as a solution, suspension, or emulsion. Alternatively,the pharmaceutical compositions can be administered by inhalation,intravenously, or intramuscularly as a liposomal suspension. In someembodiments, the pharmaceutical composition is formulated for oraladministration. In other embodiments, the pharmaceutical composition isformulated for intravenous administration.

In some embodiments of a pharmaceutical composition comprising aneffective amount of the presently-disclosed small molecule antagonistsof the kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2)and at least one pharmaceutical excipient, the pharmaceuticalcomposition comprises one or more additional therapeutic agents, asdefined above. In certain embodiments, the one or more additionaltherapeutic compounds comprise one or more of a PFKFB3 inhibitor, aPFKFB2 inhibitor, and a PFKFB1 inhibitor.

In other embodiments of the presently-disclosed subject matter, a methodof inhibiting PFKFB4 in a cell comprising contacting the cell with aneffective amount of the presently-disclosed small molecule antagonistsof the kinase domain of PFKFB4 (including the compounds of Formulae (I),(II), and (III), (VI), (V), (VI), and (VII) as well as 5MPN or MPN-2) isprovided. In certain embodiments, PFKFB4 is specifically inhibited bythe presently-disclosed small molecule antagonists of the kinase domainof PFKFB4. Thus, in certain embodiments, the presently-disclosed smallmolecule antagonists of the kinase domain of PFKFB4 are specificinhibitors of the activity of PFKFB4. In some aspects, the specificinhibitors of PFKFB4 inhibits the activity of PFKFB4 by at least about25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% 99%, or any value or range in between, but does not inhibit theactivity of PFKFB1, PFKFB2, PFKFB3 by more than about 20%, 15%, 10%, 5%,2%, or 1%.

In some embodiments of a method of inhibiting PFKFB4 in a cellcomprising contacting the cell with an effective amount of thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (VI),(V), (VI), and (VII) as well as 5MPN or MPN-2), the cell is a mammaliancell. In certain embodiments, the cell is a cancer cell. In otherembodiments, the cell is derived from a cell line comprising H460,H1299, H441, H522. A549, MDA-MB-231, LNCaP, HCT116, or LLC cell lines.

In other embodiments of the presently-disclosed subject matter, a methodof inhibiting PFKFB4 in a subject in need thereof comprisingadministering to the subject an effective amount of thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2) is provided. In certainembodiments, the presently-disclosed small molecule antagonists of thekinase domain of PFKFB4 (including the compounds of Formulae (I), (II),and (111), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) isadministered at a dosage effective for specifically inhibiting PFKFB4.In some aspects, the specific inhibitors of PFKFB4 inhibits the activityof PFKFB4 by at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%. 75%, 80%, 85%, 90%, 95% 99%, or any value or range in between, butdoes not inhibit the activity of PFKFB1, PFKFB2, PFKFB3 by more thanabout 20%, 15%, 10%, 5%, 2%, or 1%.

In some embodiments of a method of inhibiting PFKFB4 in a subject inneed thereof comprising administering to the subject an effective amountof the presently-disclosed small molecule antagonists of the kinasedomain of PFKFB4 (including the compounds of Formulae (I), (II), and(III), as well as 5MPN or MPN-2), with said small molecule antagonistsadministered orally. In other embodiments, the presently-disclosed smallmolecule antagonists of the kinase domain of PFKFB4 (including thecompounds of Formulae (I), (II), and (III), (IV), (V), (VI), and (VII)as well as 5MPN or MPN-2) are administered intravenously.

In some embodiments of a method of inhibiting PFKFB4 in a subjectcomprising administering to the subject an effective amount of thepresently-disclosed small molecule antagonists of the kinase domain ofPFKFB4 (including the compounds of Formulae (I), (II), and (III), (IV),(V), (VI), and (VII) as well as 5MPN or MPN-2), the subject remainssubstantially free of signs of toxicity.

In other embodiments of the presently-disclosed subject matter, a methodof reducing proliferative capacity of a cell comprising contacting thecell with an effective amount of the presently-disclosed small moleculeantagonists of the kinase domain of PFKFB4 (including the compounds ofFormulae (I), (II), and (III), (IV), (V), (VI), and (VII) as well as5MPN or MPN-2) is provided. In certain embodiments, the cell iscontacted with the presently-disclosed small molecule antagonists of thekinase domain of PFKFB4 (including the compounds of Formulae (I), (II),and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2) at adosage effective for specifically inhibiting PFKFB4. In some aspects,the specific inhibitors of PFKFB4 inhibits the activity of PFKFB4 by atleast about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. 75%, 80%,85%, 90%, 95% 99%, or any value or range in between, but does notinhibit the activity of PFKFB1, PFKFB2, PFKFB3 by more than about 20%,15%, 10%, 5%, 2%, or 1%.

In some embodiments of a method of method of reducing proliferativecapacity of a cell comprising contacting the cell with an effectiveamount of the presently-disclosed small molecule antagonists of thekinase domain of PFKFB4 (including the compounds of Formulae (I), (II),and (III), (IV), (V), (VI), and (VII) as well as 5MPN or MPN-2), thecell is a mammalian cell. In certain embodiments, the cell is a cancercell. In other embodiments, the cell is derived from a cell linecomprising H460, H1299. H441, H522, A549, MDA-MB-231, LNCaP, HCT116, orLLC cell lines.

In other embodiments of the presently-disclosed subject matter, a methodof reducing fructose-2,6-bisphosphate (F2,6BP) in a cell comprisingcontacting the cell with an effective amount of the presently-disclosedsmall molecule antagonists of the kinase domain of PFKFB4 (including thecompounds of Formulae (I), (II), and (III), (IV), (V), (VI), and (VII)as well as 5MPN or MPN-2) is provided. In some embodiments, the cell isa mammalian cell. In certain embodiments, the cell is a cancer cell.

EXAMPLES

The following examples are given by way of illustration and are in noway intended to limit the scope of the present invention.

Example 1 Material and Methods

Cell lines and cell culture: H460, H1299, H441, H522 and A549 non-smallcell lung cancer (NSCLC), MDA-MB-231 (breast), LNCaP (prostatic) andHCT116 (colon) adenocarcinoma and Lewis lung carcinoma (LLC) cell lineswere obtained from ATCC and used within 6 months of acquisition, PFKFB4⁷ear pinna Fibroblasts isolated from TamCrc/loxP/PFKFB4^(−/−) mice wereimmortalized as described previously (Chesney J et al.Fructose-2,6-bisphosphatc synthesis by6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) isrequired for the glycolytic response to hypoxia and tumor growth.Oncotarget2014 Aug. 30; 5(16):6670-86). Normal bronchial epithelialcells (NHBE) were obtained from Lonza and NHBE cells expressingtelomerase, SV40 large T antigen and activated Ras (hT/LT/Ras) were agift from Dr. B. J. Rollins, Dana Farber Cancer Institute. All celllines were tested and found negative for mycoplasma (PCR MycoplasmaDetection Kit, ABM). Cell lines were grown in DMEM (A549, LNCaP,MDA-MB-231, LLC and PFKFB4^(−/−)), RPMI 1640 (H460, H1299, H441, H522)and McCoy's 5A media (HCT116) (all from Invitrogen) containing 10% fetalcalf serum (Hyclone). NHBE and hT/LT/Ras cells were grown in BEGMcontaining SingleQuots (Lonza). All lines were cultured at 37° C. in 5%CO₂. In certain experiments, 4-hydroxytamoxifen (4HT, Sigma-Aldrich) wasadded to PFKFB4^(−/−) fibroblasts at indicated concentrations.

Cell Viability: Cells were incubated in 20% trypan blue (Sigma) for 5minutes. Cells excluding trypan blue were counted using a standardhemocytometer (Hausser Scientific) to determine total numbers of viablecells. Data are expressed as mean±SD of three experiments.

PFKFB4 Modeling and Compound Screen: The PFKFB4 homology model used therat testes PFKFB4 isozyme X-ray structure (PDB code 1BIF) as astructural template. An alignment was generated using Clustal W (ChennaR el al. Multiple sequence alignment with the Clustal series ofprograms. Nucleic Acids Res2003 Jul. 1; 31(13):3497-500). Four homologymodels were generated using Modeller (Sali A. Blundell T L. Comparativeprotein modeling by satisfaction of spatial restraints. J Mol Biol 1993Dec. 5; 234(3):779-815), and the structure that best reproduced thePFKFB4 binding site selected for further use. The residues essential toligand binding and protein activity for PFKFB4 were correlated toequivalent residue numbers in the consensus structure. The catalyticsite residues were selected to produce a residue-based protomol forSurflex 1.33 (Jain A N. Surflex: fully automatic flexible moleculardocking using a molecular similarity-based search engine. J Med Chem2003Feb. 13:46(4):499-511) for the virtual screening run using the 2007ZINC-drug-like library containing 3,381,225 compounds. Thehighest-scoring 100 molecules were identified for purchase. Allcomputational work, and virtual screening was done in the Brown CancerCenter Molecular Modeling Facility. The top 30 commercially availablecompounds were purchased and examined for inhibitory effects on H460cell proliferation and recombinant PFKFB4 activity.

Transfections: For siRNA experiments, cells growing in 6-well plateswere transfected with control (Stealth Negative Control Medium GC,Invitrogen) or PFKFB4 siRNA (siFB4, HSS107863, Invitrogen) usingLipofectamine RNAiMax (Invitrogen) and harvested as indicated. Foroverexpression experiments, cells were transfected with pCMV-XL4(vector) or pCMV-XL4 containing full-length PFKFB4 (Origene) usingLipofectamine 2000 (Invitrogen) and harvested as indicated.

Protein extraction and Western blotting: Protein extraction and blottingwere conducted as previously described. Membranes were probed withantibodies to PFKFB4 (Abeam) or β-actin (Sigma) followed byHRP-conjugated goat anti-rabbit or anti-mouse secondary antibodiesrespectively (Pierce). Data shown are representative of threeexperiments.

Kinase assays: The fructose-6-phosphate kinase activity of humanrecombinant PFKFB4 in the presence of DMSO±indicated 5MPN concentrationswas assayed as previously described. The activity of 5MPN against 97kinases was examined using a commercially available active-sitedependent competition binding assay core service (KINOMEscanEDGE) thatquantifies the capacity of lest agents to compete with an immobilized,active-site directed ligand using a DNA-tagged kinase and immobilizedligand and compound. For example, enzyme inhibition was studies using aADP-Glo assay. The effect of MPN-2 on the activity of recombinant humanPFKFB4 enzyme was examined in an ADP-Glo assay (Promega), which measuresADP formed from a kinase reaction. ADP is converted into ATP, which isconverted into light by a luciferase. PFKFB4 protein was exposed toMPN-2 at indicated concentrations in the presence of ATP andfructose-6-phosphate and following manufacturer's instructions, ADP-Gloand kinase detections reagents were added. The data are shown as thedecrease in luminescence caused by MPN-2 relative to DMSO.

F2,6BP measurements: Cells or tissues were prepared as previouslydescribed and F2,6BP content measured using a coupled enzyme reactionfollowing the method of Van Schaftingen et at (Van Schaftingen E et al.A kinetic study of pyrophosphate: fructose-6-phosphatephosphotransferase from potato tubers. Application to a microassay offructose 2,6-bisphosphate. Eur J Biochem1982 December; 129(1): 191-5)and normalized to total cellular protein measured by the bicinchoninicacid assay (BCA, Thermo Scientific). For example, H460 cells wereexposed to the indicated concentrations of MPN-2. In order to measureF2,6BP, cells were harvested, washed with PBS, lysed in NaOH/Trisacetate by heating at 80° C. and lysates neutralized to pH 7.2. F2,6BPcontent was measured using a coupled enzyme reaction following themethod of Van Schaftingen et al (Eur J Biochem. 1982; 129(1): 191-195).The F2,6BP concentration was normalized to total cellular proteinmeasured by the bicinchoninic acid assay (BCA, Thermo Scientific) Alldata are expressed as the mean±SD of three experiments. Statisticalsignificance was assessed by the two-sample t test (independentvariable).

Glycolysis Assay: H460 cells were exposed to the indicatedconcentrations of MPN-2 and glycolysis production was examined. Cellsgrowing in 6-well plates were incubated in 500 μl of complete mediumcontaining 1 μCi of 5-[³H]glucose per well for 60 min in 5% CO₂ at 37°C. Media was collected, ³H₂O formed via glycolysis from the5-[³H]glucose measured and counts normalized as previously described.For example, the medium was then collected and centrifuged to pellet anysuspended cells. To separate the ³H₂O formed via glycolysis from the5-[³H]glucose added to the medium, an evaporation technique in a sealedsystem was utilized. Briefly, 150 μl aliquots of medium were added toopen tubes that were placed upright inside scintillation vialscontaining 1 ml of H₂O. The scintillation vials were sealed, and the³H₂O produced by glycolysis through enolase and released to the mediumwas allowed to equilibrate with the H₂O in the outer vial for 48 h at37° C. The amounts of ³H₂O that had diffused into the surrounding H₂Owas measured on a Tri-Carb 2910 liquid scintillation analyzer (PerkinElmer) and compared with ³H₂O and 5-[³H]glucose standards. Proteinconcentration was determined using the BCA assay and counts werenormalized to protein concentration. All data are expressed as themean±SD of three experiments. Statistical significance was assessed bythe two-sample t test (independent variable).

ATP Measurements: H460 cells were exposed to the indicatedconcentrations of MPN-2 and ATP production was examined. Cells werelysed and intracellular ATP determined as described previously. Forexample, cells were washed (while still adherent) with cold PBS 1×,lysed with Passive Lysis Buffer (1×; Molecular Probes, Invitrogen) addeddirectly to the plates, and immediately harvested by scraping. Thelysates were flash frozen (to −80° C.) and thawed (to 37° C.) once toaccomplish complete lysis and then centrifuged (at 4° C.) for 30 secondsto clear the lysates. Intracellular ATP levels were determined using abioluminescence assay (Molecular Probes), utilizing recombinant fireflyluciferase and its substrate, D-luciferin. The luminescence was read ina TD-20/20 luminomeler (Turner Designs) at 560 nm. The ATP values werecalculated using an ATP standard curve. The protein concentrations ofthe lysates were estimated using the BCA assay (Pierce Biotechnology)and ATP was expressed as pmol/μg protein. All data are expressed as themean±SD of three experiments. Statistical significance was assessed bythe two-sample t test (independent variable).

Proliferation Assays: H460 non-small cell lung cancer cells were platedin 24 well plates and exposed to increasing concentrations of theindicated inhibitors of Formula IV (FBR1-02 corresponds to

land FBR1-09 corresponds to

DMSO was used as vehicle. After 24, 48 and 72 hours of exposure, cellswere detached and viable cells were counted by Trypan blue exclusion(representative counts at 48 hours shown).

Flow Cytometry: To measure apoptosis, cells were stained with annexin Vand propidium iodide and examined as previously described. For example,H460 cells were exposed to increasing concentrations of the PFKFB4inhibitor (MPN-2), the PFKFB3 inhibitor PFK15 (Tocris) or both andviable cells were counted at 24, 48 and 72 hours. DMSO used as vehicle.For cell cycle experiments, cells were detached, washed with cold PBSand fixed in 70% ethanol (4° C. 30 minutes). The cells then werepelleted by centrifugation, resuspended in PBS containing PI and RNaseA, incubated at 37° C. in the dark for 30 minutes and analyzed by flowcytometry (BD FACSCalibur). Data were analyzed using FlowJo software(TREE STAR Inc.). Results were calculated as the mean±SD of threeexperiments. The effects (Fa values) of the series of concentrations ofMPN-2 and PFK-15 were examined individually and in combination at aconstant ratio of 1:1. From these data, the CI values at various Falevels were calculated (using the CompuSyn program and the CI algorithm,from Chou and Martin) and are also represented as a Combination Indexplot and an isobologram for the combination of MPN-2 and PFK15. The CIvalues are <1 indicating that the combination of MPN-2 and PFK15 issynergistic (also seen in both plots by location of the data pointsbelow the lines that indicate additive effects).

In vivo Studies. The pharmacokinetic profile was determined in femaleC57BL/6 mice following IV and oral administration of 5MPN. Using onlyfemale mice lowered the animal numbers required for meaningful resultswithout issues of potential gender differences in exposure. Eight timepoints (n=3/time point) were used to determine PK parameters calculatedusing WinNonLin v5.0. Plasma samples were extracted using acetonitrileand analyzed by LC/MS-MS using a PhenomexSynergi Polar-RP 4 micron50×2.0 mm column eluted with a biphasic mobile phase (0.5% formic acidin acetonitrile and water).

For xenograft studies, exponentially growing LLC or H460 cells weredetached, washed and resuspended in PBS. Female C57BL/6 mice (JacksonLabs) were injected with LLC cells (n=10/group, s.c., 1×10⁶ cells) andfemale BALB/c athymic mice (Jackson Labs) with H460 cells (n=10/group,s.c, 5×10⁶ cells). Tumor masses were determined in a blinded fashionwith Vernier calipers using the formula: mass (mg)=(width, mm)²×(length,mm)/2 as previously described. When tumor masses were 150-200 mg, micewere randomized to daily intraperitoneal DMSO or 5MPN at the indicateddose in 50 μL DMSO. Tumor measurements and body weights were followeddaily. All data are expressed as the mean±SD of two experiments.Statistical significance was assessed by the two-sample t test(independent variable).

At the end of the experiment, animals were euthanized, tumors removedand sections fixed in 10% formaldehyde for immunohistochemistry orsnap-frozen in liquid nitrogen for analyses. Subsets of tumor-bearingmice (n=3) were injected i.p. with 2-[¹⁸F]-fluoro-2-deoxyglucose (FDG,150 μCi. 100 μl) and imaged by micro-positron emission tomography aspreviously described. Regions of interest in the tumors and cerebellumwere quantified in quadruplicate and expressed as the mean±SD of thetumor:cerebellar FDG uptake ratio. Animal experiments were approved bythe University of Louisville Institutional Animal Care and UseCommittee.

Immunohistochemistry: Formalin-fixed, paraffin-embedded tissue sectionswere processed as previously described then incubated with anti-Ki-67primary antibody (Abeam) overnight, followed by HRP-linked goatanti-rabbit secondary antibody (1:300. Pierce). Sections were developedwith 3,3′-diaminobenzidine tetrahydrochloride, counterstained, scannedand analyzed with the positive pixel count algorithm as previouslydescribed. Data are depicted as % positive pixcels/total pixels ±SD.

Example 2 Small Molecule Antagonists of PFKFB4

Results: We utilized the X-ray structure of the Ratus norvegicus testesPFKFB4 (Hasemann C A et al. The crystal structure of the bifunctionalenzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealsdistinct domain homologies. Structure 1996 Sep. 15; 4(9): 1017-29.) toconduct an in silico screen of >18 million small molecules to identifypotential compounds that may interact with the fructose 6-phosphate(F6P) binding domain of PFKFB4. Over one hundred compounds wereidentified, scored, ranked, and analyzed based on their associationpotential with the active site within PFKFB4. We physically tested the30 best-score compounds for their ability to inhibit the kinase activityof recombinant PFKFB4. For example,5-[(8-methoxyquinolin-4-yl)amino]pentyl nitrate (termed 5MPN; FIG. 1Aand FIG. 1B), significantly inhibited PFKFB4 activity (FIG. 1C). Basedon Lineweaver-Burk analyses, this compound appears to be a competitiveinhibitor of the F6P binding site (FIG. 1D). Importantly, this compounddid not inhibit PFK-1 or PFKFB3 which share the identical substrate andare co-cxpressed with PFKFB4 in multiple cell lines and required forglucose metabolism (no inhibition of kinase activity with 10 μM).Additionally, a panel of 97 protein kinases was not inhibited by 10 μMof 5MPN providing further support for the selectivity of this compoundfor PFKFB4 (KINOMEscan, data not shown).

Example 3 Pharmacological Inhibition of PFKFB4 by 5MPN is SelectivelyCytostatic to Transformed Cells

Results: H460 cells are lung adenocarcinoma cells that harbor severalcommon oncogenic mutations (CDKN2A^(del457), KRAS^(Q61H).PIK3CA^(E545k), STR11^(Q37X)) and are sensitive to inhibition of PFKFB4using siRNA molecules. We examined the anti-metabolic effects of 5MPN onH460 cells and found that this agent reduced the intracellularconcentration of F2,6BP, glycolysis and ATP (FIG. 2A) which in turnresulted in a reduction in cell proliferation (FIG. 2B). We alsoexamined the effect of 5MPN on the proliferation of non-small cell lungcancer (H460, H1299, H441, H522 and A549), breast adenocarcinoma(MDA-MB-231), prostatic adenocarcinoma (LNCaP) and colon adenocarcinoma(HCT116) cell lines and observed a dose-dependent reduction in growthover 48 hours (FIG. 2C). Given that PFKFB4 has been found to beexpressed by normal lung epithelia, we next examined the relativeeffects of 5MPN on normal human bronchial epithelial (NHBE) cells versusNHBE cells that had been sequentially immortalized with telomerase andlarge T antigen and transformed with H-Ras^(V12) (hT/LT/Ras cells). Wefound that the NHBE cells were virtually unaffected whereas hT/LT/Rascell growth was suppressed similar to other transformed cells (FIG. 2D).In order to interrogate the requirement of PFKFB4 inhibition for theobserved suppression of proliferation (on-target effects), we nextexamined the effects of genetic modulation of PFKFB4 on theanti-proliferative effects of 5MPN. We found that whereasover-expression of PFKFB4 protected H460 cells from 5MPN, genomicdeletion of Pfkfb4 sensitized cells to 5MPN (FIG. 2E and FIG. 2F), thussupporting the concept that inhibition of PFKFB4 by 5MPN is causing theobserved reduction in H460 cell proliferation. Taken together, thesedata indicate that 5MPN is a potent inhibitor of PFKFB4 that selectivelysuppresses the proliferation of transformed cells.

Example 4 PFKFB4 Inhibition with 5PMN Causes a G1 Cell Cycle Arrest ThatIs Reversed by PFKFB4 Over-Expression

Results: We noted a marked reduction in viable H460 cells after exposureto 5MPN for 48 hours (see FIG. 2B) and postulated that 5MPN was inducingapoptosis, arresting cell cycle progression, or both. Whereas weobserved only a minimal increase in apoptotic cells after 5MPN exposureor selective PFKFB4 siRNA transfection (FIG. 3A and FIG. 3B), wcobserved a marked G1 arrest with both 5MPN and PFKFB4 siRNA (FIG. 3C andFIG. 3D). We then over-expressed PFKFB4 and exposed the H460 cells to5MPN at the indicated concentrations and assessed the effects on cellcycle and F2,6BP. We found that over-expression of PFKFB4 reversed thereduction in F2,6BP (FIG. 3E) and G1 arrest (FIG. 3F) caused by 5MPN.These studies suggest that 5MPN is suppressing PFKFB4 which in turn isresulting in a reduction in the G1/S transition.

Example 5 5MPN has High Oral Bioavailability and Suppresses the GlucoseUptake and Growth of Tumors in Mice

Results: The pharmacokinetics of intravenous and oral administration of5MPN was studied and revealed that both routes were adequate to achievepotentially therapeutic concentrations when administered daily (FIG. 4Aand FIG. 4B). Given the potential usefulness of oral administration interms of cost and convenience, we elected to pursue this route insubsequent toxicity and efficacy pre-clinical studies. Initially, wedosed C57BL/6 mice with 120 mg/kg PO for two weeks and analyzed theeffect on complete blood counts, electrolytes, hepatic and renalfunction, body mass and the gross and histological appearance of thebrain, heart, lungs, liver, kidneys and spleen. We found no signs oftoxicity either from these objective measures or from any behavioral orclinical changes (i.e. ruffled fur, lethargy, ataxia or laboredrespiration). Importantly, at this oral dose, it was determine that 5MPNsuppressed the growth of Lewis lung carcinomas grown in syngeneic mice(FIG. 4C) and H460 human lung adenocarcinoma xenografts grown in athymicmice (FIG. 4J) without affecting body weight (FIG. 4D and FIG. 4K).Next, the effects of oral administration of 5MPN on intratumoral F2,6BPand glucose uptake by LLC xenografts was examined. There was a markedreduction in F2,6BP (FIG. 4E) and 2-[¹⁸F]-fluoro-2-deoxyglucose uptakeusing positron emission tomography (FIG. 4F). It was confirmed that 5MPNcaused a G1 arrest in LLC cells in vitro similar to H460 cells (FIG.4G). The number of Ki67-positive cells was examined since Ki-67expression correlates with later S and G2 phases of the cell cycle(Sasaki K et al. The cell cycle associated change of the Ki-67 reactivenuclear antigen expression. J Cell Physiol 1987 December;133(3):579-84). It was determined that oral administration of 5MPNcaused a reduction in Ki67-positive cells in the LLC xenografts (FIG. 4Hand FIG. 4I) suggesting that 5MPN may be reducing cell cycle progressionin vivo.

Example 6 MPN-2 Inhibits Recombinant PFKFB4 Enzyme Activity, Decreasesthe Production of F2,6BP, Decreased Proliferation of Cancer Cells, andhas High Oral Bioavailability

Results: 5-[(8-methoxy-2-methylquinolin-1-ium-4-yl)amino]pentyl nitrate(MPN-2) significantly inhibited PFKFB4 activity (FIG. 5B) and F2,6BPproduction (FIG. 5C). MPN-2 decreased the proliferation of a humancancer cell line, H460 NSCLC (FIG. 5D). Viable cells counted at 48 hoursand at 72 hours. Pharmacokinetic analysis of intravenous and oraladministration of MPN-2 determined that MPN-2 exhibits high oralbioavailability. FIG. 5E shows the oral and intravenous pharmacokineticproperties of MPN-2 in CD-1 mice (N=3)

Example 7 MPN-2 Causes Decreased Proliferation of Cancer Cells Precededby a Reduction in Intracellular Glycolysis and ATP, DecreasedProliferation of Cancer Cells, and Tumor Growth in Mice

Results: MPN-2 significantly decreased intracellular glycolysis (FIG.6A) and ATP (FIG. 6B) production of H460 NSCLC cells treated withDMSO±the indicated concentrations of MPN-2. The effects on glycolysis(FIG. 6A) and ATP (FIG. 6B) were measured after 48 hours. Furthermore,MPN-2 significantly decreased the proliferation of various cancer celllines. FIG. 6C shows the decreased proliferation of MCF7, SK-BR-3, H460,and A549 cells exposed to DMSO±the indicated concentrations of MPN-2after 24-72 hours (72 hours shown). Furthermore, it was determined thatMPN-2 suppressed the growth of Lewis lung carcinomas grown in C57BL/6mice (FIG. 6D). When tumors reached a mass of 150-200 mg, mice wererandomized to daily administration of DMSO or 5MPN in DMSO at theindicated dose by intraperitoneal injection (10 mg/kg, for eleven days).

Example 8 Formula IV Compounds Decreased Proliferation of Cancer Cells

Results: We also examined the effect of the compounds of Formula IV onthe proliferation of H460 non-small cell lung cancer, H460. Formula IVcompounds, specifically

significantly decreased the proliferation of the H460 NSCLC cells (FIG.7).

Example 9 Dual PFKFB4 and PFKFB3 Inhibition with MPN-2 and PFK15,Respectively, Causes a Synergistic Increase in Cell Death

Results: Studies conducted by the instant investigators have revealedthat PFKFB4 expression is increased by PFKFB3 inhibition, suggestingthat PFKFB4 may compensate for decreased PFKFB3 expression and activityand, importantly, may limit the efficacy of PFKFB3 inhibitors, includingPFK15 (Tocris). Thus, we also examined the effect of combination therapyof a PFKFB4 inhibitor and a PKFB3 inhibitor. FIG. 8 shows thatsimultaneous administration of MPN-2 (determined by the instantinvestigators to be a PFKFB4 inhibitor) and PFK15 (Tocris, acommercially available PFKFB3 inhibitor) synergistically increased celldeath in vitro. Thus, combination therapy with PFK15 (and other PFKFB3inhibitors) and the presently-disclosed small molecule antagonists ofthe kinase domain of PFKFB4 may be used to provide an effectivechemotherapeutic regimen.

Example 10 Synthesis of the Small Molecule Antagonists of the KinaseDomain of PFKFB4 (Including Various Compounds of Formulae (I), (II), and(III), (IV), (V), (VI), and (VII))

Referring to FIG. 9, the synthesis of the instantly disclosed smallmolecule antagonists of the kinase domain of PFKFB4 (including variouscompounds of Formulae (I), (II), and (III), (VI), (V), (VI), and (VII)began with the condensation of Meldrum's acid (1) with aniline 2 in thepresence of trimethyl orthoformate to afford adduct 3 in excellent yield(Dutta, A. K.; et al. Discovery of4-(4-(2-((5-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)ethyl)piperazin-1-yl)quinolin-8-oland Its Analogues as Highly Potent Dopamine D2/D3 Agonists and as IronChelator: In Vivo Activity Indicates Potential Application inSymptomatic and Neuroprotective Therapy for Parkinson's Disease. J. MedChem. 2010.53.2114-2125). Cyclization was facilitated by heating 3 indiphenyl ether to produce 8-methoxyquinolin-4-ol (4) core, which wassubsequently converted to chloride 5 by refluxing in phosphoryl chloride(Id.). The carbon linker was installed by heating chloride 5 with acommercially available primary amine (Perez, B. C.; et al.N-Cinnamoylated Chloroquine Analogues as Dual-Stage Antimalarial Leads.J. Med Chem. 2013.56, 556-567). In the final step, several conditionswere surveyed for nitric acid ester formation and many were sluggish orproduced multiple byproducts. Ultimately, treating alcohol 6 withconcentrated nitric acid, acetic acid, and acetic anhydride in ethylacetate produced the desired product (Kourounakis, P. N. et al. Nitricoxide releasing derivatives of tolfenamic acid with anti-inflammatoryactivity and safe gastrointestinal profile. Bioorg. Med Chem. 2005, 13,6485-6492.)

Still referring to FIG. 9, the synthesis is amendable to produce alibrary of derivatives. For example, the carbon linker can be altered bytreating chloride 5 with a variety of nucleophiles. Furthermore, theprimary alcohol of compound 6 can be functionalized with groups otherthan the nitric acid ester. Attempts were made to prepare the2-methylquinoline salt by treating 7 with methyl iodide in acetonitrile.Decomposition was observed. Tt is hypothesized that the salt may need tobe prepared prior to the installation of the nitric acid ester.

All documents cited are incorporated herein by reference; the citationof any document is not to be construed as an admission that it is priorart with respect to the present invention

1.-264. (canceled)
 265. A method of treating cancer in a subject in needof treatment thereof, the method comprising administering to the subjectan effective amount of a compound selected from Formula (IV):

wherein: n is 1-6; R₁ is carboxylic acid, methyl sulfamide, carboxylicacid methyl ester, hydroxide, nitrate, or tert-butyl carbamate; R₂ canbe present or absent, and when present is a C₁-C₅ alkyl; R₃ can bepresent or absent, and when present is a C₁-C₅ alkoxy, chloride, orhydrogen; and wherein if R₂ is present and located on the nitrogen ofthe quinoline group, then the nitrogen has a positive charge.
 266. Themethod of claim 265, wherein the subject is substantially free of signsof toxicity.
 267. The method of claim 265, further comprisingadministering to the subject one or more additional therapeuticcompounds and the one or more additional therapeutic compounds comprisesone or more of a PFKFB3 inhibitor, a PFKFB2 inhibitor, and a PFKFB1inhibitor.
 268. The method of claim 265, wherein n=5, R₁ is nitrate, R₃is methoxy, or a combination thereof.
 269. The method of claim 265,wherein n=5, R₁ is nitrate, and R₃ is methoxy.
 270. The method of claim265, wherein the compound is 5MPN or MPN-2.
 271. A method of inhibitingPFKFB4 in a subject, the method comprising administering to the subjectan effective amount of a compound selected from Formula (IV):

wherein: n is 1-6; R₁ is carboxylic acid, methyl sulfamide, carboxylicacid methyl ester, hydroxide, nitrate, or tert-butyl carbamate; R₂ canbe present or absent, and when present is a C₁-C₅ alkyl; R₃ can bepresent or absent, and when present is a C₁-C₅ alkoxy, chloride, orhydrogen; and wherein if R₂ is present and located on the nitrogen ofthe quinoline group, then the nitrogen has a positive charge.
 272. Themethod of claim 271, wherein the compound of Formula (IV) isadministered at a dosage effective for specifically inhibiting PFKFB4.273. The method of claim 271, wherein the compound of Formula (IV) isadministered orally or intravenously.
 274. The method of claim 271,wherein the subject remains substantially free of signs of toxicity.275. The method of claim 271, wherein n=5, R₁ is nitrate, R₃ is methoxy,or a combination thereof.
 276. The method of claim 271, wherein n=5, R₁is nitrate, and R₃ is methoxy.
 277. The method of claim 271, wherein thecompound is 5MPN or MPN-2.
 278. A pharmaceutical composition comprisingan effective amount of a compound selected from Formula (IV)

wherein: n is 1-6; R₁ is carboxylic acid, methyl sulfamide, carboxylicacid methyl ester, hydroxide, nitrate, or tert-butyl carbamate; R₂ canbe present or absent, and when present is a C₁-C₅ alkyl; R₃ can bepresent or absent, and when present is a C₁-C₅ alkoxy, chloride, orhydrogen; and wherein if R₂ is present and located on the nitrogen ofthe quinoline group, then the nitrogen has a positive charge; and atleast one pharmaceutical excipient.
 279. The pharmaceutical compositionof claim 278, wherein an effective amount of the compound is an amountthat specifically inhibits PFKFB4.
 280. The pharmaceutical compositionof claim 278, wherein the pharmaceutical composition is formulated fororal administration or is formulated for intravenous administration.281. The pharmaceutical composition of claim 278, wherein n=5, R₁ isnitrate, R₃ is methoxy, or a combination thereof.
 282. Thepharmaceutical composition of claim 278, wherein n=5, R₁ is nitrate, andR₃ is methoxy.
 283. The pharmaceutical composition of claim 278, whereinthe compound is selected from the group consisting of:


284. The pharmaceutical composition of claim 278, wherein the compoundis 5MPN or MPN-2.